Isolation and Structural Studies of Sulfated Glycoproteins of Hog Gastric Mucosa

Sulfated glycoproteins were purified from tryptic digests of hog stomach mucosa. The sulfated glycoprotein fractions were isolated by ethanol and cetylpyridinium chloride precipitation followed by free-flow electrophoresis. The separation of the sulfated glycoproteins was performed by elution from DEAE-Sephadex A-50 with NaCl. After gel filtration of the major fractions on Bio-Gel A-50m, final separation was achieved by rechromatography on DEAE-Sephadex. The homogeneity of the purified fractions was confirmed by electrophoresis on cellulose acetate strips at pH 1.76 and 9.0. All of the purified sulfated glycoprotein fractions exhibited strong (A + H) blood-group activity. The extent of activity depended on the sulfate content and decreased slightly as a sulfate content increased. Two groups of similar but distinct carbohydrate chains were separated, one containing 18 and the other 14 glycosyl units. The carbohydrate composition of the studied fractions was found to be (in residues per 2 residues of N-acetylgalactosamine): fucose, 1.9 to 4.1; galactose, 5.9 to 7.9; and N-acetylglucosamine, 3.7 to 4.0. The sulfate was present in each fraction with a molar ratio of hexosamine to sulfate ranging from 2.4 to 5.4. N-acetylglucosamine 6-sulfate was the only sulfated sugar found in the studied fractions. The carbohydrate chains of the sulfated glycoproteins from hog stomach mucosa are linked to the protein through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Sequential Smith degradation, partial acid hydrolysis of the native and partially degraded glycoproteins, enzymatic digestion, and immunological assays established the carbohydrate sequence and some of the linkages between the sugars.